Elucidating the contributions of ubiquitin ligase complexes to endoplasmic reticulum associated degradation (ERAD)
Lead Research Organisation:
University of Oxford
Department Name: Ludwig Institute for Cancer Research
Abstract
Proteins are the molecular machines of cells, performing a wide range of essential functions. A broad range of inherited human diseases can be traced back to single proteins that do not fold correctly. A change in the genetic code results in a protein that is unable to reach the correct 3-dimensional structure necessary to function. Other disorders are associated with conditions within cells that are unsuitable for proteins in general to fold correctly. For those proteins that are secreted or expressed on the surface of cells, protein folding occurs in the intracellular compartment known as the endoplasmic reticulum (ER). Within the ER, each protein is evaluated by quality control mechanisms that decide whether it's folded correctly. When proteins misfold, a complex remediation scheme is engaged which removes those forms from the ER and destroys them. Collectively, this mechanism is known as ER-associated degradation (ERAD). As cell surface and secreted proteins represent approximately 30% of all the human proteins made, distinguishing and degrading such a wide range of potentially misfolded proteins presents a significant challenge. ERAD uses a network of interconnected factors able to identify, target, deliver and degrade non-native proteins with various configurations. A central component in this scheme is the family of ubiquitin ligases embedded in the ER membrane. One example is Hrd1, which forms nexuses of ERAD activity by scaffolding different accessory factors on both sides of the ER membrane. This MRC project aims to characterise both the composition and organisation of Hrd1-containing complexes and to ascertain the contributions that the entire family of ER-resident ubiquitin ligases make to ERAD.
The benefits of the proposed research will be to advance our understanding of how misfolded proteins are degraded in cells, and how this fundamental mechanism alleviates cellular stress that is the hallmark of many diseases. Our current view of ERAD has been based on a limited analysis of individual components and substrates. The proposed research aims to advance this picture by 1) characterising in detail, the functional Hrd1 complexes the carry out ERAD, 2) using a proven isolation strategy and mass spectrometry to characterise other ER-resident ubiquitin ligase complexes that participate in ERAD and 3) determining how cells use expression of the different ERAD components to respond to stress. As the number of diseases linked to protein misfolding grows, it is anticipated that the comprehensive analysis proposed here will advance our understanding of how these conditions may arise. Moreover, this study may help direct efforts to devise novel and alternative therapeutic strategies for the treatment of disorders of associated with protein misfolding and trafficking, such as cystic fibrosis and alpha1-antitrypsin deficiency.
The benefits of the proposed research will be to advance our understanding of how misfolded proteins are degraded in cells, and how this fundamental mechanism alleviates cellular stress that is the hallmark of many diseases. Our current view of ERAD has been based on a limited analysis of individual components and substrates. The proposed research aims to advance this picture by 1) characterising in detail, the functional Hrd1 complexes the carry out ERAD, 2) using a proven isolation strategy and mass spectrometry to characterise other ER-resident ubiquitin ligase complexes that participate in ERAD and 3) determining how cells use expression of the different ERAD components to respond to stress. As the number of diseases linked to protein misfolding grows, it is anticipated that the comprehensive analysis proposed here will advance our understanding of how these conditions may arise. Moreover, this study may help direct efforts to devise novel and alternative therapeutic strategies for the treatment of disorders of associated with protein misfolding and trafficking, such as cystic fibrosis and alpha1-antitrypsin deficiency.
Technical Summary
ERAD represents the principal remediation mechanism for non-native, maturation-incompetent proteins whose unabated accumulation can disrupt optimal ER function. ERAD employs a network of accessory factors localised to both sides the ER membrane that coordinate with the ubiquitin-proteasome system (UPS) to degrade misfolded integral membrane and secreted proteins. Membrane embedded ubiquitin ligases (E3s) such as Hrd1 form nexuses for ERAD activity by assembling macromolecular complexes in the ER along the transmembrane axis. This proposal aims to identify the individual components and domains essential for formation of Hrd1-containing complexes using velocity sedimentation to monitor size, rationally designed truncations/deletions to map the interaction domains, RNAi (and possibly TALEN-based genomic editing) to determine essential accessory factors and radiolabeling-densitometry to ascertain stoichiometry. This proposal also draws on a proven affinity purification-LC-MS/MS strategy to establish the interaction networks (and potentially substrates) of the 26 ER-resident E3s putatively involved in ERAD. Quantitative analysis using the Nanostring hybridization platform will be used to track changes gene expression of components in the ERAD network (with a focus on ER-resident E3s and their interactors) that result in response to ER stress conditions. This study will provide the first comprehensive analysis of ER-resident E3 complexes, their organisation and their expression with the goal of ultimately determining the contributions they individually make to the ERAD capacity and its ability to restore ER functionality during periods of stress-induced global misfolding.
Planned Impact
It is anticipated that this research will provide an immediate impact for the community of research scientists and health professionals by providing critical insights into the general mechanisms by which ERAD attains the capacity to recognise, deliver, target and degrade non-native proteins. Our work focuses on characterising the molecular components and regulation of ER-resident ubiquitin ligase complexes with the long-term view to determine how they collectively function to exercise constitutive ERAD activity as well as respond to provide the enhanced capacity needed during times when cells experience ER stress. This research will be of significant interest to both academic and industrial biologists both in the UK and abroad whose interests lie in protein quality control, protein folding diseases and conditions characterised by ER stress. Those researchers and clinicians specialising in protein folding disorders will find the data generated in the study of particular interest, as it will help to further define the molecular mechanisms governing the fate of their protein of interest. Moreover, the strategy proposed herein will lay the groundwork for future studies that will define the molecular rules governing how and why misfolded proteins require particular complexes for degradation. While the research plan presented in this proposal is unlikely to result in any directly patentable or otherwise commercially exploitable material in the near future, it may indirectly lead to the development of novel therapeutic strategies to treat protein misfolding disorders in the years following the conclusion of the project.
The impact of our work on society and economics here in the UK will be in the potential of these studies to identify novel molecular targets for therapies against protein folding disease such as cystic fibrosis, alpha1-antitrypsin disease and lysosomal storage diseases. In the future, our work may prompt pharmaceutical and biotech companies to target components within protein quality control mechanisms and the ERAD pathway in particular, to treat these and other diseases. Moreover, the role of ERAD as an alleviator of ER stress may have potential in therapeutics, as ER stress is increasingly being appreciated as a phenomenon associated with cancer, obesity, diabetes and conditions such as irritable bowel syndrome. If realised, the above examples would provide economic benefits locally and contribute to the improvement in quality of life for individuals in the UK, as well as abroad.
The impact of our work on society and economics here in the UK will be in the potential of these studies to identify novel molecular targets for therapies against protein folding disease such as cystic fibrosis, alpha1-antitrypsin disease and lysosomal storage diseases. In the future, our work may prompt pharmaceutical and biotech companies to target components within protein quality control mechanisms and the ERAD pathway in particular, to treat these and other diseases. Moreover, the role of ERAD as an alleviator of ER stress may have potential in therapeutics, as ER stress is increasingly being appreciated as a phenomenon associated with cancer, obesity, diabetes and conditions such as irritable bowel syndrome. If realised, the above examples would provide economic benefits locally and contribute to the improvement in quality of life for individuals in the UK, as well as abroad.
People |
ORCID iD |
John Christianson (Principal Investigator) |
Publications
Anzilotti C
(2019)
An essential role for the Zn2+ transporter ZIP7 in B cell development.
in Nature immunology
Christianson JC
(2014)
Cleaning up in the endoplasmic reticulum: ubiquitin in charge.
in Nature structural & molecular biology
Schulz J
(2017)
Conserved cytoplasmic domains promote Hrd1 ubiquitin ligase complex formation for ER-associated degradation (ERAD).
in Journal of cell science
Tang HY
(2014)
EDEM2 and OS-9 are required for ER-associated degradation of non-glycosylated sonic hedgehog.
in PloS one
Glaeser K
(2018)
ERAD-dependent control of the Wnt secretory factor Evi.
in The EMBO journal
Vitale M
(2019)
Inadequate BiP availability defines endoplasmic reticulum stress.
in eLife
Volkmar N
(2016)
New MAPS for misfolded proteins.
in Nature cell biology
Dersh D
(2014)
OS-9 facilitates turnover of nonnative GRP94 marked by hyperglycosylation.
in Molecular biology of the cell
Raducu M
(2016)
SCF (Fbxl17) ubiquitylation of Sufu regulates Hedgehog signaling and medulloblastoma development.
in The EMBO journal
Elliott PR
(2016)
SPATA2 Links CYLD to LUBAC, Activates CYLD, and Controls LUBAC Signaling.
in Molecular cell
Volkmar N
(2018)
The ER membrane protein complex (EMC) promotes biogenesis of sterol-related enzymes maintaining cholesterol homeostasis
in Journal of Cell Science
Guna A
(2018)
The ER membrane protein complex is a transmembrane domain insertase.
in Science (New York, N.Y.)
Higgins R
(2015)
The Unfolded Protein Response Triggers Site-Specific Regulatory Ubiquitylation of 40S Ribosomal Proteins.
in Molecular cell
Title | Interaction mapping of endoplasmic reticulum ubiquitin ligases identifies modulators of innate immune signalling |
Description | Ubiquitin ligases (E3s) embedded in the endoplasmic reticulum (ER) membrane regulate essential cellular activities including protein quality control, calcium flux, and sterol homeostasis. At least 25 different, transmembrane domain (TMD)-containing E3s are predicted to be ER-localised, but for most their organisation and cellular roles remain poorly defined. Using a comparative proteomic workflow,... |
Type Of Material | Database/Collection of data |
Year Produced | 2020 |
Provided To Others? | Yes |
Impact | Identification of a novel ER-resident ubiquitination complex regulating the innate immune signalling through STING. Revealed the broad interaction profiles of ER-resident E3 enzymes which will help researchers uncover their cellular functions. |
URL | https://www.ebi.ac.uk/pride/archive/projects/PXD019559 |
Description | COST Proteostasis |
Organisation | European Cooperation in Science and Technology (COST) |
Country | Belgium |
Sector | Public |
PI Contribution | attendance at scientific conference |
Collaborator Contribution | organise scientific meetings, courses and exchanges |
Impact | presentation at 1st Proteostasis Meeting |
Start Year | 2014 |
Description | Kessler Lab Collaboration |
Organisation | University of Oxford |
Department | Target Discovery Institute (TDI) |
Country | United Kingdom |
Sector | Academic/University |
PI Contribution | Our group provided the samples for proteomic analysis |
Collaborator Contribution | Our collaborators processed our proteomic samples and assisted us with its analysis and refinement of the datasets |
Impact | DOI: 10.7554/eLife.57306 |
Start Year | 2020 |
Description | Conference invitation - Paris, France |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invitation to present work in at International Symposium on Endoplasmic Reticulum in Paris, France. Presented findings to >100 researchers in a hybrid presentation style, prompting a Q&A period following the talk and discussions afterwards. |
Year(s) Of Engagement Activity | 2021 |
URL | https://itbcde.aviesan.fr/er-meeting-2021.html |
Description | EMBO Girona 2014 - poster |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | poster presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | discussed results with other scientists fruitful discussions on research topic |
Year(s) Of Engagement Activity | 2014 |
URL | http://events.embo.org/14-er/ |
Description | FASEB Ubiquitin Meeting |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | poster presentation |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | discussed data with other researchers met new researchers in my field |
Year(s) Of Engagement Activity | 2014 |
URL | https://secure.faseb.org/FASEB/meetings/summrconf/Programs/11619.pdf |
Description | Podcast (University of Oxford) |
Form Of Engagement Activity | Engagement focused website, blog or social media channel |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Public/other audiences |
Results and Impact | Participated in filming of podcast for the Nuffield Department of Medicine. The purpose of this activity was to disseminate information about the ongoing research in our lab to the general public and local researchers |
Year(s) Of Engagement Activity | 2016 |
URL | http://www.ndm.ox.ac.uk/john-christianson-cleaning-up-misfolded-proteins |
Description | Proteostasis Meeting Valencia 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Type Of Presentation | keynote/invited speaker |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | interest sparked from several audience members met new people in related field of ubiquitin biology |
Year(s) Of Engagement Activity | 2014 |
URL | http://proteostasis-2014.cipf.es/ |
Description | School visit (Cokethorpe School) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Local |
Primary Audience | Schools |
Results and Impact | Participated in career day at local secondary school where I gave a short presentation about careers in biomedical science, which sparked questions and discussion afterwards. |
Year(s) Of Engagement Activity | 2015 |
URL | http://www.cokethorpe.org.uk/media/news/article/1548/Careers-Awareness-Talks |
Description | Scientific Conference - Proteostasis (Lisbon, Portugal) |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Invited speaker at conference with leading European experts on Proteostasis. Questions and discussions followed. |
Year(s) Of Engagement Activity | 2016 |
URL | http://cost-proteostasis.eu/blog/event/proteostasis-and-its-biological-implications-management-commi... |
Description | Scientific meeting - EMBO ER conference |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | International |
Primary Audience | Professional Practitioners |
Results and Impact | Selected presentation at conference attended by world's leading experts in the ER. Questions and discussion followed. |
Year(s) Of Engagement Activity | 2016 |
URL | http://events.embo.org/16-er/ |
Description | Thames Valley Cancer Network - Myeloma & Plasma Cell Dycrasias Meeting 2014 |
Form Of Engagement Activity | A talk or presentation |
Part Of Official Scheme? | No |
Geographic Reach | Regional |
Primary Audience | Health professionals |
Results and Impact | brief discussions of implications of our work improved connections with specialists in field |
Year(s) Of Engagement Activity | 2014 |